The present invention relates to a process for making the cyclic oligopeptide, cyclosporin A, and, more particularly, to a microbiological method using a new producer strain to make and isolate cyclosporin A.
Cyclosporin A is a member of a group of cyclic undecapeptides with antiinflammatory, immunosuppressive, antifungal and antiparasitic properties. The immunomodulatory properties are of particular interest in medicine, since they are important in treatments of transplantation surgery and for autoimmune diseases.
Cyclosporins are a new class of compounds, which were first described in Swiss Patent 589,716 and 603,790. They have also been reviewed in the Monograph by Borel (Cyclosporin, Progress in Allergy 38, Karger Press, 1986). The chemical structure and biological-pharmacological properties were described in the latter Monograph.
In the production method for Cyclosporin A, the ability of microorganisms for synthesis of the naturally occurring Cyclosporins is used, since one cultivates certain species of microorganisms as producing agents in a fermentation process using the standard culturing conditions in a nutrient solution, which contains a complex substrate. Similarly in a way, which is generally known, one isolates the desired product and purifies it.
In extensive screening experiments numerous fungus species were found to have cyclosporin-forming properties. DREYFUSS (Sydowia, Bd. 39, 1986, pp. 22 to 36) describes an exclusively emersed Cyclosporin-forming capability for Cylindrocarpon and Fusarium fungi, while Cyclosporin formation was detected in submersed cultures of Hyphae fungi, Tolypocladium geodes, Trichoderma viride, Neocosmospora vasinfecta, Isaria felina Verticillium spec., Acremonium spec. and Beauveria nivea (originally Tolypocladium inflatum). The highest productivity can be obtained with strains of the fungi, Beauveria nivea. This strain is of particularly significance for the large scale production of Cyclosporin A. According to the description by TRABER et al (in J.Antibiotics, Vol. 42, 1989, pp. 591 to 597) a mutant of Beauveria nivea (in the cited article described as a mutant of Tolypocladium inflatum NRRL 8044) produces about 500 mg of Cyclosporin A per liter. In a chemical synthetic medium containing malic acid and sucrose and ammonium as nitrogen source KOBEL and TRABER (Europ. J. Appl. Microbiol.Biotechnol., vol. 14, 1982, pp.237-240) obtain a yield of 101 mg Cyclosporin A per liter in 14 culture days. By feeding with select aminoacids in a concentration of 8 g/l, the yield level could be improved, but this method is however in no way relevant for an economic industrial production method. Similar disadvantages are present in the fermentation method of AGATHOS et al (Ann.N.Y. Acad.Sci. 506, 1987, pp. 657-662), which describes a fermentation in a semisynthetic medium with a yield of 530 mg cyclosporin A per liter after 16 culture days. However, in this method the use of maltose leads to a high rate of operating expense in an industrial process.
Regarding the economics of using a producing strain, the described volume-time-yields for an industrial process are exceptionally unsatisfactory, which causes other disadvantages in the isolation and purification of different cyclosporins, particularly cyclosporin A, connected necessarily with loss of agent. The described methods for isolation of Cyclosporin A from a culture suspension of different strains must consider in all cases the expected sensitivity of the peptide bond in the Cyclosporin-A molecule and begins by avoiding higher temperatures with a liquid-liquid extraction of the culture suspension or with an extraction of the separated wet mycelium (U.S. Pat. No. 4,117,118; U.S. Pat. No. 4,215,199; German Patent 2 455 859; Belgian Patent 866 810; v. Wartburg et al, Helv.Chim.Acta 59,(1976), p. 1075). The extraction residue is usually distributed between 30% methanol and petroleum ether and the material with fat removed is repeatedly chromatographed on silica gel, aluminum oxide and/or Sephadex LH 20, on which a considerable adsorption occur, which proceeds up to 1000-fold of the substrate(German Patent 2 455 859). This extensive purification work and the stress of immediate work-up of the producing culture media with wet mycelium are substantial difficulties for the known production methods. Of particular disadvantage in the known methods is the high cost of the solvents used, particularly because they are not environmentally friendly. Also environmental considerations must come into play in their disposal.